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Abstract: Although dynorphins are widely involved in the control of not only nociceptive neurotransmission but also a variety of brain functions such as memory and emotion, no natural regulator for inducing the mRNA expression of prodynorphin (Pdyn), a precursor protein of dynorphins, is known. Using primary cultures of rat cortical neurons, we found that pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the vasoactive intestinal polypeptide (VIP)/secretin/glucagon neuropeptide family, markedly induces Pdyn mRNA expression. PACAP was much more effective than VIP, indicating a major role for PAC1 in the PACAP-induced Pdyn mRNA expression. The increase in Pdyn mRNA expression was independent of de novo protein synthesis. Administration of forskolin, an activator for adenylate cyclase/protein kinase A (PKA), but not TPA, an activator for protein kinase C (PKC), induced Pdyn mRNA expression, suggesting a major role for PKA. The involvement of PKA was supported by the inhibition of PACAP-induced Pdyn mRNA expression upon addition of H89, an inhibitor for PKA. The PACAP-induced potentiation of NMDA-R was involved in the mRNA expression of Bdnf or c-fos but not Pdyn. These results suggest PACAP to be an upstream regulator for inducing Pdyn mRNA expression through PKA. [ABSTRACT FROM AUTHOR]

Background: During microRNA (miRNA) maturation in humans and flies, Drosha and Dicer cut the precursor transcript, thereby producing a short RNA duplex. One strand of this duplex becomes a functional component of the RNA-Induced Silencing Complex (RISC), while the other is eliminated. While thermodynamic asymmetry of the duplex ends appears to play a decisive role in the strand selection process, the details of the selection mechanism are not yet understood. Results: Here, we assess miRNA strand selection bias in humans and fruit flies by analyzing the sequence composition and relative expression levels of the two strands of the precursor duplex in these species. We find that the sequence elements associated with preferential miRNA strand selection and/or rejection differ between the two species. Further, we identify another feature that distinguishes human and fly miRNA processing machinery: the relative accuracy of the Drosha and Dicer enzymes. Conclusion: Our result provides clues to the mechanistic aspects of miRNA strand selection in humans and other mammals. Further, it indicates that human and fly miRNA processing pathways are more distinct than currently recognized. Finally, the observed strand selection determinants are instrumental in the rational design of efficient miRNA-based expression regulators. [ABSTRACT FROM AUTHOR]

Alternative splicing is a sophisticated nuclear process that regulates gene expression. It represents an important mechanism for enhancing the functional diversity of proteins. Our current knowledge of alternatively spliced variants is derived mainly from mRNA transcripts, and very little is known about their protein tertiary structures. We carried out a large-scale analysis of known alternatively spliced variants at both protein sequence and structure levels and have shown that threading is, in general, a viable approach for modeling structures of alternatively spliced variants. An examination of alternative splicing at the protein sequence level revealed that the size of splicing events follows the power law distribution and the majority of splicing isoforms harbor only one or two alternations. We examined alternative splicing in the context of protein 3D structures and found that the boundaries of alternative splicing events generally happen in coil regions of secondary structures and exposed residues and the majority of the sequences involved in splicing are located on the surface of proteins. In light of these findings, we then proceeded to demonstrate that threading represents a useful tool for structure prediction of alternative splicing isoforms and addressed the fold stability issue of threading-based structure prediction by molecular dynamics simulation. Our analysis and the insights gained have helped to establish a viable method for structure prediction of alternatively spliced isoforms at the genome scale. [ABSTRACT FROM AUTHOR]